CN113728875A

CN113728875A - Method for cultivating wild reed mushrooms in batches and application

Info

Publication number: CN113728875A
Application number: CN202111139694.3A
Authority: CN
Inventors: 刘西周
Original assignee: Tianjin Normal University
Current assignee: Tianjin University; Tianjin Normal University
Priority date: 2021-09-28
Filing date: 2021-09-28
Publication date: 2021-12-03

Abstract

The invention relates to a method for cultivating wild reed mushrooms in batches and application thereof, which comprises the steps of mother seed preparation, stock seed preparation, cultivated seed preparation, batch cultivation culture material preparation, stock induction condition establishment and fruiting body batch cultivation mode optimization. The method can be used for artificial batch cultivation of wild reed mushrooms, and has the advantages of low production cost, simple operation, large primordia generation quantity, high fruiting body quality, capability of realizing batch cultivation and the like. The fruiting body of the bulrush mushroom cultivated by the method adopts a tray type fruiting, and the growth period is 45 days. The method provides powerful support for protecting species diversity of wetland ecosystem, saving endangered wild mushroom germplasm resources and realizing reasonable and orderly development and utilization of wild reed mushrooms, and the artificial batch cultivation method of the wild reed mushrooms has extremely high development and popularization values and remarkable economic and ecological benefits.

Description

Method for cultivating wild reed mushrooms in batches and application

Technical Field

The invention belongs to the field of research on artificial domestication and mushroom cultivation of wild edible fungi, and relates to a batch cultivation method and application of wild reed mushrooms, in particular to a method for obtaining fruiting bodies by batch cultivation of the wild reed mushrooms.

Background

The Qili sea wetland in Tianjin is a national level wetland natural protection area, is located in the northeast Ninghe river area in Tianjin, has a total area of about 95 square kilometers, is internally provided with wild mushroom which is called as the Phragmites communis, has unique taste, rich nutrition and delicious and unique taste, is deeply favored by local people, and is generated in thick humus after hot weather in summer and continuous overcast and rainy weather due to scarcity and high price. In recent years, as the ecological environment of the Qili sea is destroyed, water sources are gradually exhausted, the wetland is degraded, more and more wetlands are filled for agriculture or construction, the reed area is reduced, and in addition, artificial over-picking and special growth environment thereof need to grow under the action of common environmental factors such as specific temperature, humidity, illumination and soil, the yield and quality of wild reed mushrooms in the Tianjin Qili wetland are reduced year by combining various environmental and human factors, according to 2016 (7 months) -up to now through comprehensive tracking investigation and statistics of local folk environmental protection people, the wild reed mushrooms in the area are difficult to find, and the polar face the risk of extinction, the inventor applies for a method for artificially cultivating the wild reed mushrooms and an invention patent of the application in 2017, the invention mainly solves the problem that if mushrooms can be produced in a small test, proves that the artificial domestication can normally form sporocarp in a laboratory in a small test range, but mass production still has uncertainty. Subsequent continuous experiments find that basic conditions and methods for cultivating the reed mushrooms in batches can be obtained through optimization.

At present, through data examination and reading, no report related to the success of batch artificial domestication is found, and the situation facing the extinct is further aggravated undoubtedly. Therefore, in order to protect species diversity of the wetland ecosystem in the field, save and protect endangered wild mushroom germplasm resources and realize sustainable, reasonable and ordered development and utilization of the Tianjin Qihai wild reed mushrooms, the invention establishes a batch artificial cultivation mode, provides powerful support for protecting species diversity of the wetland ecosystem in the field, saving endangered wild mushroom germplasm resources, carrying out deep research in the later period and realizing reasonable and ordered development and utilization of the Tianjin Qihai wild reed mushrooms, and the artificial batch cultivation mode of the Tianjin Qihai wild reed mushrooms has great development prospect and extremely remarkable ecological and economic benefits.

Disclosure of Invention

The artificial cultivation mode of wild reed mushrooms in the Tianjin Qilihai comprises mother seed preparation, stock seed preparation, cultivated seed preparation, batch cultivation culture material preparation, stock induction condition establishment and fruiting body batch cultivation mode optimization.

The invention is realized by the following measures:

a method for artificial batch cultivation of wild reed mushrooms is characterized by comprising the following steps

(1) Reed mushroom mother seed production and culture condition

Optimized culture medium of 10 g/L of glucose, 200g/L of potato (leaching liquor) and MgSO₄·7H₂O 1.5 g/L，KH₂PO₄3 g/L, 20 g/L agar powder and VB₁0.01 g/L, 2.5 g/L of straw powder, 25 g/L of straw (leaching liquor) and 5 g/L of reed humus, inoculating a wild reed mushroom strain to a plate culture medium in a sterile operation manner, sealing the plate culture medium by using a sealing film after inoculation, and culturing at the constant temperature of 29 ℃ for 10 days to obtain light yellow sparse colonies, namely a mother strain pure culture; the pH value suitable for the growth of the mother strain mycelium is 5-7; the growth temperature range of the mycelium is suitable to be 19-34 ℃; culturing at 29 deg.C under constant temperature and dark condition for 15-20 days to obtain 90mm plate; the wild reed mushroom strain has a nucleotide sequence shown in SEQ ID NO. 1;

(2) preparation of stock seed

Reed humus collected from the habitat of wild reed mushrooms is filtered by a screen to remove impurities, and the reed humus is obtained. Soaking the wheat grains for 24 h. Adding appropriate amount of water, and cooking until the seed coat is not broken. And (5) draining the water. Weighing the materials according to the formula (90% of reed humus +10% of wheat grains, water content of 60%), mixing, immediately filling into polypropylene cultivation bags (300 mm × 150mm × 0.05 mm), and sterilizing at 121 deg.C for 2 h. After sterilization, the stock culture bags were transferred to a sterile room and the inoculation was performed 1cm in a clean bench after cooling to 32=37 ℃²Inoculating 3 pieces of mother seeds, culturing at 29 deg.C in dark at constant temperature, and growing full bags in 23-25 days;

(3) production of cultivars

Taking 2-4cm of fresh mildew-free long rice straw sections, soaking the fresh mildew-free long rice straw sections in 3% lime water for 12h for pre-wetting, draining, and controlling the water content to be 60% -65%. The method for processing and preparing the wheat grain and the reed humus is the same as that of the original seed. Weighing materials according to a formula (52% of straw, 24% of wheat grains, 24% of reed humus and 65% of water content), mixing the materials, filling the materials into a polypropylene cultivation bag (300 mm multiplied by 150mm multiplied by 0.05 mm), bagging 150g of wet materials, and sterilizing for 2 hours at 121 ℃. After cooling, inoculating the stock seeds in each bag in a super clean bench, culturing at the constant temperature of 29 ℃ in dark with the humidity of 65 percent and growing the bags full in 25-28 days;

(4) shallow tray type mushroom cultivation material preparation

Inoculating a pure culture of the reed mushroom cultivated species full of flat plates into a mushroom cultivation culture material polypropylene tray under the aseptic condition, wherein the tray specification is as follows: 320mm multiplied by 240mm multiplied by 90mm, the formula of the wild reed mushroom cultivation compost is 52% of straw, 20% of wheat bran, 24% of reed humus, 4% of vermiculite, the water content is 65%, and the pH is natural; filling about 600g of wet material in each dish, and sterilizing for 2h at 121 ℃;

(5) spawn running of shallow plate type mushroom cultivation compost: cooling, inoculating proper amount of cultivar in a clean bench, culturing at 29 deg.C under constant temperature and dark condition with humidity of 65% for 25-28 days;

(6) primordium induction conditions: culturing the plectania carotovora mycelium at 25 deg.C for 12h, then culturing at 15 deg.C for 12h, and stimulating at 10 deg.C for 10 days to obtain large amount of primordium;

(7) culturing conditions of sporocarp: culturing a mushroom-cultivating tray which is transformed by primordia and has a large amount of primordia at the constant temperature of 25 ℃, wherein the humidity is 85-90%, the 1000-1500lx scattered light is used, the illumination date is 12h/24h, and the fruiting bodies can be obtained after culturing for 10-12 days.

The artificial cultivation method of the invention comprises the following steps of obtaining the morphological characteristics description of the reed mushroom fruiting body by artificial cultivation: the fruiting body is thick, soft and light in meat quality, the diameter of the pileus is 5-10cm, the pileus is light yellow, the pileus is smooth and does not have scales, the meat quality is concave in the center, the edge becomes wavy after the fruiting body is matured, the edge of the pileus waxiness is sharp, the pileus is spread and distributed sparsely, the pileus tends to be widened, the position of the stipe is mesogenic, aseptic rings and a bacteria support, and the spores are printed white after the fruiting body is matured.

The molecular biological characteristics of the bulrush fruiting body obtained by artificial cultivation are described as follows:

the PCR amplification ITS sequence fragment is extracted after genome DNA of a bulrush fruiting body obtained by artificial cultivation is extracted, BLAST search is carried out in a GenBank nucleic acid sequence database to obtain ITS sequences of a plurality of species with higher homology, similarity comparison is carried out, and the mushroom is identified as one mushroom of Helicoverpa (Hygrophopsidae) and Helicoverpa (Hygrophoroproppsis) according to the comparison result and has the nucleotide sequence shown in SEQ ID NO. 1.

The invention further discloses a method for artificially cultivating wild reed mushrooms in batches and application of the method for artificially cultivating the reed mushrooms in the Tianjin Qili sea area. The experimental results show that: by adopting the cultivation method disclosed by the invention, the fruiting bodies of wild reed mushrooms in Tianjin Qihai can be obtained in batches after about 45 days, and powerful support is provided for species diversity of a wetland ecosystem in Tianjin Qihai, saving endangered wild mushroom germplasm resources, carrying out deep research in the later period and realizing reasonable and ordered development and utilization of wild reed mushrooms in Tianjin Qihai.

The invention is described in more detail below:

the fruiting body obtained by artificial batch cultivation of wild Phragmites communis in Tianjin Qili sea is characterized in that: the pileus is about 5-10cm in size, light yellow, smooth in pileus surface, without scales, semi-waxy or fleshy, and sunken in center; the fungus folds are usually waxy, growing and sparse; the spore is white, the stipe is 2-3cm long, the fungus grows, the fungus is medium and solid, the fungus is not a ring and a fungus tray, the fungus cover is rolled after the fungus is mature, and the edge is wavelike and curly.

1. Preparation of mother seed and its culture condition

Optimized culture medium of 10 g/L of glucose, 200g/L of potato (leaching liquor) and MgSO₄·7H₂O 1.5 g/L，KH₂PO₄3 g/L, 20 g/L agar powder and VB₁0.01 g/L, 2.5 g/L of straw powder, 25 g/L of straw (leaching liquor) and 5 g/L of reed humus, selecting a pure culture of pleomogenous hypha on a slant of the reed mushroom, quickly inoculating the pure culture to a plate culture medium in a super clean workbench, sealing the pure culture in a sealing film at 29 ℃ for 10 days to obtain light yellow sparse colonies, namely a pure culture of a mother strain; the pH value suitable for the growth of the mother strain mycelium is 5-7; the growth temperature range of the mycelium is suitable to be 19-34 ℃; culturing at 29 deg.C under constant temperature and dark condition for 15-20 days to obtain 90mm plate;

2. preparation of stock seed

Reed humus collected from the habitat of wild reed mushrooms is filtered by a screen to remove impurities, and the reed humus is obtained. Soaking the wheat grains for 24 h. Adding appropriate amount of water, and cooking until the seed coat is not broken. And (5) draining the water. Weighing materials according to a formula (90% of reed humus +10% of wheat grains, water content of 60%), mixing the materials, immediately filling into polypropylene cultivation bags (300 mm multiplied by 150mm multiplied by 0.05 mm), and sterilizing at 121 DEG CAnd (5) sterilizing for 2 h. After sterilization, transferring the stock culture bag to a sterile room, cooling to 32-37 deg.C, inoculating 1cm in a super clean bench²Inoculating 3 pieces of mother seeds, culturing at 29 deg.C in dark at constant temperature, and growing full bags in 23-25 days;

3. production of cultivars

Taking 2-4cm of fresh mildew-free long rice straw sections, soaking the fresh mildew-free long rice straw sections in 3% lime water for 12h for pre-wetting, draining, and controlling the water content to be 60-65%. The method for processing and preparing the wheat grain and the reed humus is the same as that of the original seed. Weighing materials according to a formula (52% of straw, 24% of wheat grains, 24% of reed humus and 65% of water content), mixing the materials, filling the materials into a polypropylene cultivation bag (300 mm multiplied by 150mm multiplied by 0.05 mm), bagging 150g of wet materials, and sterilizing for 2 hours at 121 ℃. Cooling, inoculating appropriate amount of stock seeds in each bag in a superclean bench, culturing at 29 deg.C under constant temperature and dark condition with humidity of 65%, and filling the bags after 23-25 days;

4. shallow tray type mushroom cultivation material preparation

Inoculating a pure culture of the reed mushroom cultivated species full of flat plates into a mushroom cultivation culture material polypropylene tray under the aseptic condition, wherein the tray specification is as follows: 320mm multiplied by 240mm multiplied by 90mm, the formula of the wild reed mushroom cultivation compost is 52% of straw, 20% of wheat bran, 24% of reed humus, 4% of vermiculite, the water content is 65%, and the pH is natural; filling about 600g of wet material in each dish, and sterilizing for 2h at 121 ℃; spawn running of shallow plate type mushroom cultivation compost: cooling, inoculating proper amount of cultivar in a clean bench, culturing at 29 deg.C under constant temperature and dark condition with humidity of 65% for 23-25 days;

5. primordial induction conditions

Culturing the plectania carotovora mycelium at 25 deg.C for 12h, then culturing at 15 deg.C for 12h, and stimulating at 10 deg.C for 10 days to obtain large amount of primordium;

6. batch culture condition of sporophore

The fruiting shallow tray with a large amount of primordia is cultured for 10 days under the constant temperature condition of 25 ℃, the humidity is 85-90%, 1000-1500lx scattered light and the illumination date is 12h/24h to obtain mature fruiting bodies.

7. The fruit body obtained in the present invention was evaluated as follows:

(1) morphological feature description of reed mushroom fruiting body obtained by artificial batch cultivation

The method is mainly characterized in that the sporocarp obtained by artificial batch cultivation is soft, the diameter of the pileus is 5-10cm, the pileus is light yellow, the pileus is smooth and has no scale and meat, the center is sunken, the edge is wavy after the pileus is mature, the edge of the pileus is sharp, the pileus is extended, sparsely distributed and tends to widen towards the pileus, the stipe is middle-growing, aseptic rings and fungus trays, and spores are printed white after the pileus is mature;

(2) description of molecular biology characteristics of fruiting bodies of reed mushrooms obtained by artificial batch cultivation

Artificially cultured and obtained bulrush fruiting body, extracting genome DNA of the bulrush fruiting body, performing PCR amplification on ITS sequence fragments, performing BLAST search in a GenBank nucleic acid sequence database to obtain ITS sequences of a plurality of species with high homology, and performing similarity comparison, wherein the comparison result shows that the bulrush fruiting body is identified as a mushroom of Helicoverpa (Hygrophoroppsidae) and Helicoverpa (Hygrophoroppsis). Has a nucleotide sequence shown as SEQ ID NO. 1

(3) The manual batch cultivation of the Qili sea wild reed mushroom fruiting bodies has the advantages of strong taste, delicious taste and strong fragrance.

(4) Comparing the shape of wild bulrush with that of artificially domesticated bulrush

The results show that: the fruiting bodies obtained by artificial domestication and batch cultivation are slightly small, the mushroom flesh is thick, the color is light yellow, the leather quality degree is low, and the commercial value is high.

The artificial cultivation method of the wild reed mushrooms disclosed by the invention has the positive effects that:

(1) the method can be used for artificial cultivation of wild reed mushrooms in the Qilihai of Tianjin, and the growth cycle of the reed mushroom fruiting bodies is about 45 days. Has the advantages of low production cost, simple operation, large primordia generation quantity, capability of obtaining a batch of sporocarp and the like.

(2) The establishment of the artificial cultivation mode of the wild reed mushrooms in Tianjin Qihai has extremely high values in saving, protecting, researching, developing and popularizing the special endangered edible fungi reed mushrooms in Tianjin Qihai.

(3) The method provides powerful support for protecting species diversity of wetland ecosystem, saving endangered wild mushroom germplasm resources and realizing reasonable and orderly development and utilization of wild reed mushrooms, and the artificial batch cultivation method of the wild reed mushrooms has extremely high development and popularization values and remarkable economic and ecological benefits.

Drawings

FIG. 1 artificial batch cultivation to obtain fruiting body.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available. The wild Phragmites communis is picked from Tianjin Qili sea.

Example 1

The invention adopts a tissue separation method to obtain strains, selects partial sporocarp of wild mushroom fungus which has normal color and is not invaded by pests, removes impurities on the surface, wipes the surface with 75 percent alcohol, longitudinally cuts the partial sporocarp for separation into two parts under the aseptic condition, takes tissue blocks with the size of mung bean at the junction of stipe pileus, quickly inserts the tissue blocks into a PDA flat plate by using tweezers, and carries out dark culture in a constant temperature incubator at 29 ℃ for 10-15 days for later use.

Example 2

Bag content gradient single factor experiment

Based on an optimal culture material formula (52% of straw, 24% of wheat grains and 24% of reed humus), according to a previous test, taking 150g of wet material per bag as reference, respectively setting 6 gradients of wet weight of the bag material of 100 g/bag, 150 g/bag, 200 g/bag, 250 g/bag, 300 g/bag and 350 g/bag, treating 10 bags each, respectively inoculating an equal amount of test cultivars, culturing at a constant temperature of a 29 ℃ incubator, and determining the optimal bag filling amount according to the growth conditions of hypha and primordium.

The result shows that the bagging weight of 200g is the proper bagging amount for the screened bulrush mushroom artificial cultivation.

TABLE 1 influence of different bag contents on the growth of hypha and primordium of Phragmitis mushroom

Note: the representative hyphae does not germinate, the (+) represents that the hyphae is weak in growth vigor, the (+) represents that the hyphae is general in growth vigor, the (+) represents that the hyphae is excellent in growth vigor, and the (+) represents that the hyphae is vigorous in growth vigor; o stands for no primordia present, OO stands for a smaller number of primordia, and OOO stands for a larger number of primordia. (same table)

Example 3

Straw proportion gradient single-factor experiment

Based on an optimal culture material formula, according to an early test of 52% straw culture material as a reference, 6 gradients of 2% straw, 12% straw, 22% straw, 32% straw, 42% straw and 52% straw are respectively arranged, less than 100% of the gradients are supplemented by vermiculite (which is only used as a filling material and does not provide nutrition required by hypha growth), 50 bags are respectively treated, an equal amount of test cultivars are respectively inoculated, constant-temperature culture is carried out at a temperature of 29 ℃, and the optimal straw proportion is determined according to the hypha and primordium growth conditions.

The results show that: the formula of the straw with the concentration of 52% is the proportion of the screened cultivation material suitable for the artificial cultivation of the reed mushrooms.

TABLE 2 influence of different straw contents on the growth of hypha and primordium of Phragmitis mushroom

Example 4

Single factor experiment of wheat grain ratio

Based on an optimal culture material formula, according to a previous test of 24% wheat grain culture material, 6 gradients of 4% wheat grains, 8% wheat grains, 12% wheat grains, 16% wheat grains, 20% wheat grains and 24% wheat grains are respectively set, less than 100% of the gradients are supplemented by vermiculite (only serving as a filling material and not providing nutrition required by hypha growth), 50 bags are processed for each treatment, an equal amount of test cultivars are respectively inoculated, constant-temperature culture is carried out at a temperature of 29 ℃, and the optimal wheat grain proportion is determined according to the hypha and primordium growth conditions.

The results show that: 20 percent of the wheat grains are the proportion of the screened cultivation material suitable for the artificial cultivation of the reed mushrooms.

TABLE 3 influence of different wheat grain contents on the growth of hypha and primordium of Phragmitis mushroom

Example 5

Reed humus proportion gradient single-factor experiment

Based on an optimal culture medium formula, according to a previous test of 24% of reed humus culture medium as a reference, respectively setting 6 gradients of 4% of reed humus, 8% of reed humus, 12% of reed humus, 16% of reed humus, 20% of reed humus and 24% of reed humus, supplementing less than 100% of the culture medium by vermiculite (only serving as a filling material and not providing nutrition required by hypha growth), respectively treating 50 bags of the culture medium, respectively inoculating equal amounts of test cultivars, culturing at a constant temperature of a 29 ℃ incubator, and determining the optimal proportion of the reed humus according to the hypha and primordium growth conditions.

The results show that: the formula of 24 percent of the reed humus is the proportion of the screened cultivation material suitable for artificial cultivation of the reed mushrooms.

TABLE 4 influence of different Phragmites on growth of hypha and primordium of Phragmites communis

Example 6

Wheat bran-substituted wheat grain proportional gradient single-factor experiment

Because wheat grains need to be pre-cooked in advance, the operation process is relatively complex, wheat grains are replaced by wheat bran, an optimal culture material formula is used as a basis, 6 gradients of 4% of wheat bran, 8% of wheat bran, 12% of wheat bran, 16% of wheat bran, 20% of wheat bran and 24% of wheat bran are respectively set according to a previous test of 24% of wheat grain culture materials as a reference, less than 100% of the gradients are supplemented by vermiculite (only serving as a filling material and not providing nutrition required by hypha growth), 50 bags of treatment are respectively inoculated into equal amount of test cultivars, constant-temperature culture is carried out in a 29 ℃ incubator, and the optimal proportion of wheat bran is determined according to the hypha and primordium growth conditions.

The results show that: the formula is 20% of wheat bran which is suitable for the cultivation material proportion of artificial cultivation of the reed mushrooms.

TABLE 5 influence of wheat bran on the growth of hypha and primordium of Phragmites communis in the presence of wheat bran

Example 7

Soil covering and soil non-covering contrast processing

After uniform inoculation of fruiting fungus sticks, after full bags are cultured under the optimal conditions, the bags are covered with soil with the thickness of 2 cm, the water content of the soil is adjusted to be 18%, the type of the soil covering material is reed humus, 50 bags are covered with soil, 50 bags are not covered with soil, according to the optimal fruiting conditions, the unprocessed group is used as a contrast, and the primordium forming condition after soil covering treatment is contrasted.

The results show that: the covering soil has little influence on the formation of primordium, and in addition, the covering soil causes a great amount of fungus bag pollution in the test process.

TABLE 6 soil and non-soil treatment results (50 replicates)

Note: -means no germination of the hyphae, or no primordia, or no fruiting. + means that there is a rare growth of hyphae, or a rare number of primordia, or a rare fruiting. (+) means better hypha growth, or better primordia quantity, or better fruiting. (+) means the growth of hypha, or the concentration of primordia, or the growth of mushroom. (the same applies below).

Example 8

Contrast treatment of sterilization and non-sterilization of covering material

After uniform inoculation of fruiting fungus sticks, full bags are cultured under the optimal conditions, the bags are covered with soil with the thickness of 2 cm, the water content of the prepared soil is 18%, the type of the soil covering material is reed humus, the soil covering material is sterilized for 50 bags, the soil covering material is not sterilized for 50 bags, according to the optimal fruiting conditions, a non-processing group is used as a contrast, and the formation conditions of biological factors (beneficial microorganisms) on the primordia of the reed mushrooms before and after the soil covering sterilization are compared.

The results show that sterilization of the casing material has little effect on the formation of primordia, but because the casing material is sterilized, although a large amount of fungus bags are contaminated in the test process, the amount of contamination is reduced compared to fungus bags treated with casing material that has not been sterilized.

TABLE 7 results of the sterile and non-sterile treatment of casing materials

Example 9

Contrast treatment of fruiting at top and side

After the fruiting fungus sticks are inoculated in a unified mode, after the fungus sticks are cultured under the optimal conditions and fully bagged, 2 kinds of treatment of fruiting with an opening at the upper end and fruiting with a cut on the side wall are respectively carried out, 50 kinds of treatment are carried out on each group, a non-treatment group is used as a control according to the optimal fruiting conditions, and the primordium formation conditions under the fruiting treatment at the top end and the side face are compared.

The results show that: the upper end cutting opening and the side wall cutting opening have shorter fruiting period than the lower end cutting opening, but deformed mushrooms appear.

TABLE 8 Top and side fruiting treatment results

Example 10

Contrast treatment of tray type fruiting and bagged cultivation fruiting

Uniformly inoculating fruiting fungus sticks, culturing in full bags under the optimal conditions, breaking the full bags of mycelia under the aseptic conditions, putting the broken mycelia in a sterile 50X 40X 10cm tray for fruiting, covering with an oxygen-permeable and air-permeable sealing film, performing tray type fruiting, treating 50 mycelia in each group, and comparing the formation conditions of primordia under the tray type fruiting and the bag cultivation fruiting according to the optimal fruiting conditions by taking a non-treated group as a control.

The results show that: compared with bag cultivation, the tray type fruiting is short in period and less in deformed mushrooms.

TABLE 9 treatment results of tray-type fruiting and bag cultivation fruiting

Example 11

Mycelium culture and its primordium induced differentiation

The culture condition of the wild reed mushroom mother seed mycelium in Tianjin Qilihai is characterized in that the mycelium is constant at 29 ℃, dark and dark, and can grow over a 75mm flat plate after being cultured for 15 days, and the culture condition of the fruiting mushroom stick mycelium is characterized in that the mycelium is constant at 25 ℃, dark and dark, has the space humidity of 60 percent, and can grow over mushroom bags (the specification is 15 multiplied by 30 multiplied by 0.04mm polypropylene plastic bags, and the wet weight of the materials is 250 g) after being cultured for 30-35 days.

The primordium differentiation of wild Phragmites communis in Tianjin Qili sea is characterized in that mycelium of a full bag is cultured for 12h at 25 ℃, 12h at 15 ℃ and stimulated for 7-10 days at 10 ℃ with continuous temperature difference, and primordium is produced in large quantity.

Example 12

Culture of fruiting body

The cultivation of wild Phragmites communis fruiting body in Tianjin Qihai is characterized in that the fruiting body can be obtained by cultivating the fungus bag with a large amount of primordium under the condition of constant temperature of 25 ℃, humidity of 85-90% (preferably 85%), scattered light of 1000-1500lx (preferably 1500 lx) for 10-15 days (preferably 15 days). The length of the ITS sequence of Pleurotus Nebrodensis is 785base, and the test report is provided by Shanghai Biotech, Inc.

SEQUENCE LISTING

<110> university of Tianjin

<120> wild reed mushroom batch cultivation method and application

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 785

<212> DNA

<213> Artificial sequence

<400> 1

tccgtaggtg aacctgcgga aggatcatta tcgaattctt tttgagaggg ggaaagggag 60

gagcagactg ttgctggctt tttttttcga cgacggtgac gctggataac ggccgaaccg 120

ttgtgagaat gtataggcat cgtgcacgtt tgtgactttt ttttcttctg atcaatagct 180

ttctccaacc cacacctgtg cactcgttgt aggctctttg aaaaaaaagg cctatgtttt 240

tctacacaca cccaatcgta tgtctataga atgtcttttt ttacgattac tgagcgcttc 300

ttcgagaaga tggtcagtaa gtaaagttta ttacaacttt cagcaatgga tctcttggct 360

ctcgcatcga tgaagaacgc agcgaatcgc gatacgtaat gtgaattgca gattttcagt 420

gaatcatcga atctttgaac gcaccttgcg ctccttggta ttccgaggag catgcctgtt 480

tgagtgtcat taaattctca actccctttg attttgctat cgaggggggc ttggacagtg 540

ggtttgctgg cgtgttttca ataattcgtc ggctttcctt aaatgcattg gcaaaaaggc 600

gaaggcatga taacgtcttt cggtgtgata atgatctcgc cgtggatgga agtgtcctag 660

ttctcctgtg cctataaccc gtttgcgtcg ttgcttgtaa tggagtgacg atgctttttt 720

gaaccttttg acctcaaatc aggtaggact acccgctgaa cttaagcata tcaataagcg 780

gagga 785

Claims

1. A method for cultivating wild reed mushrooms in batches is characterized by comprising the following steps:

(1) reed mushroom mother seed production

Inoculating the activated reed mushroom strain to a 90mm plate culture medium in a sterile operation, sealing the inoculated reed mushroom strain by using a sealing film, and culturing at the constant temperature of 29 ℃ for 10 days; the wild reed mushroom strain has a nucleotide sequence shown in SEQ ID NO. 1;

(2) reed mushroom mother culture medium and culture conditions thereof

Glucose 10 g/L, potato 200g/L (extract), MgSO₄·7H₂O 1.5 g/L，KH₂PO₄3 g/L, 20 g/L agar powder and VB₁0.01 g/L, 2.5 g/L of straw powder, 25 g/L of straw (leaching liquor) and 5 g/L of reed humus; pH suitable for hypha growth is 5-7; the growth temperature range of the mycelium is suitable to be 19-34 ℃; culturing at 29 deg.C under constant temperature and dark condition for 15-20 days to obtain 90mm plate;

(3) reed mushroom original seed production

Reed humus collected from a wild reed mushroom habitat is filtered by a screen to remove impurities to obtain the reed humus, wheat grains are soaked for 24 hours, and water is added according to the volume of 1:1.5 for cooking;

draining water, weighing, mixing, immediately packaging into polypropylene cultivation bag of 300mm × 150mm × 0.05mm, sterilizing at 121 deg.C for 2 hr, transferring to aseptic room, cooling to 32-37 deg.C, inoculating into 1cm in ultra-clean bench²Inoculating 3 pieces of mother seeds, culturing at 29 deg.C in dark at constant temperature, and growing full bags in 25-28 days; the formula refers to: 90% of reed humus and 10% of wheat grains, and the water content is 60%;

(4) reed mushroom cultivated species production

Soaking 2-4cm of fresh mildew-free long rice straw sections in 3% lime water for 12h for pre-wetting, draining water, and controlling the water content to be 60-65%; the processing and preparation method of wheat grain and reed humus is the same as that of original seeds; weighing materials according to a formula, stirring the materials, filling the materials into polypropylene cultivation bags with the thickness of 300mm multiplied by 150mm multiplied by 0.05mm, bagging 150g of wet materials, sterilizing the materials for 2 hours at the temperature of 121 ℃, cooling the materials, inoculating 1 original seed into each bag in a super clean workbench, performing dark cultivation at the constant temperature of 29 ℃, culturing the seeds with the humidity of 65 percent, and growing the seeds in the bags in 25 to 28 days; the formula refers to: 52 percent of straw, 24 percent of wheat grain and 24 percent of reed humus, and the water content is 65 percent;

(5) preparation of reed mushroom shallow tray type mushroom cultivation compost

Inoculating a pure culture of the reed mushroom cultivated species full of flat plates into a mushroom cultivation culture material polypropylene tray under the aseptic condition, wherein the tray specification is as follows: 320mm multiplied by 240mm multiplied by 90mm, the formula of the wild reed mushroom cultivation compost is 52% of straw, 20% of wheat bran, 24% of reed humus, 4% of vermiculite, the water content is 65%, and the pH is natural; filling 600g of wet material in each tray, and sterilizing for 2 hours at 121 ℃;

(6) cultivating the reed mushroom in a shallow disc type mushroom cultivation culture material for spawn running: cooling, inoculating the cultivar in a super clean bench, culturing at 29 deg.C under constant temperature and dark conditions with humidity of 65% and growth of 25-28;

(7) and (3) reed mushroom primordium induction conditions: culturing the plectania carotovora mycelium at 25 deg.C for 12h, then culturing at 15 deg.C for 12h, and stimulating at 10 deg.C for 10 days to obtain large amount of primordium;

(8) culturing conditions of sporocarp: culturing the fungus bag which has been converted by the primordium and has a large amount of primordium at the constant temperature of 25 ℃, wherein the humidity is 85-90%, the light is scattered by 1500lx at 1000 times, the illumination time is 12h/24h, and the fruiting body can be obtained after 10-12 days of culture.

2. The batch cultivation method according to claim 1, wherein the suitable mycelium growth temperature is 29 ℃, and the cultivation is performed under constant temperature and light-shielding conditions for 15-20 days.

3. The batch cultivation method of claim 1, wherein the humus of the reed is obtained by collecting humus at the root of reed in the habitat of wild reed mushrooms, and filtering the humus with a 20-mesh screen to remove impurities, and the humus mainly provides nutrients, mineral elements and water-absorbing and air-permeable physical media for the growth of the wild reed mushrooms.

4. The batch cultivation method of claim 1, wherein the mushroom cultivation compost polypropylene tray has the following specifications: 320mm 240mm 90mm, and requires high temperature resistant polypropylene material, but is not limited to this specification.

5. The method for cultivating the wild reed mushrooms in batches according to claim 1, which is applied to improving the cultivation of the reed mushrooms in the area of the seven-mile sea of Tianjin.